Efficient scar-free knock-ins of several kilobases in plants by engineered CRISPR-Cas endonucleases.

In plants and mammals, non-homologous end-joining is the dominant pathway to repair DNA double-strand breaks, making it challenging to generate knock-in events. In this study, we identified two groups of exonucleases from the herpes virus and the bacteriophage T7 families that conferred an up to 38-fold increase in homology-directed repair frequencies when fused to Cas9/Cas12a in a tobacco mosaic virus-based transient assay in Nicotiana benthamiana. We achieved precise and scar-free insertion of several kilobases of DNA both in transient and stable transformation systems. In Arabidopsis thaliana, fusion of Cas9 to a herpes virus family exonuclease led to 10-fold higher frequencies of knock-ins in the first generation of transformants. In addition, we demonstrated stable and heritable knock-ins in wheat in 1% of the primary transformants. Taken together, our results open perspectives for the routine production of heritable knock-in and gene replacement events in plants.

Improved prime editing allows for routine predictable gene editing in Physcomitrium patens.

Efficient and precise gene editing is the gold standard of any reverse genetic study. The recently developed prime editing approach, a modified CRISPR/Cas9 [clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein] editing method, has reached the precision goal but its editing rate can be improved. We present an improved methodology that allows for routine prime editing in the model plant Physcomitrium patens, whilst exploring potential new prime editing improvements. Using a standardized protoplast transfection procedure, multiple prime editing guide RNA (pegRNA) structural and prime editor variants were evaluated targeting the APT reporter gene through direct plant selection. Together, enhancements of expression of the prime editor, modifications of the 3' extension of the pegRNA, and the addition of synonymous mutation in the reverse transcriptase template sequence of the pegRNA dramatically improve the editing rate without affecting the quality of the edits. Furthermore, we show that prime editing is amenable to edit a gene of interest through indirect selection, as demonstrated by the generation of a Ppdek10 mutant. Additionally, we determine that a plant retrotransposon reverse transcriptase enables prime editing. Finally, we show for the first time the possibility of performing prime editing with two independently coded peptides.

A novel binary pesticidal protein from Chryseobacterium arthrosphaerae controls western corn rootworm by a different mode of action to existing commercial pesticidal proteins.

The western corn rootworm (WCR) Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae) remains one of the economically most important pests of maize (Zea mays) due to its adaptive capabilities to pest management options. This includes the ability to develop resistance to some of the commercial pesticidal proteins originating from different strains of Bacillus thuringiensis. Although urgently needed, the discovery of new, environmentally safe agents with new modes of action is a challenge. In this study we report the discovery of a new family of binary pesticidal proteins isolated from several Chryseobacterium species. These novel binary proteins, referred to as GDI0005A and GDI0006A, produced as recombinant proteins, prevent growth and increase mortality of WCR larvae, as does the bacteria. These effects were found both in susceptible and resistant WCR colonies to Cry3Bb1 and Cry34Ab1/Cry35Ab1 (reassigned Gpp34Ab1/Tpp35Ab1). This suggests GDI0005A and GDI0006A may not share the same binding sites as those commercially deployed proteins and thereby possess a new mode of action. This paves the way towards the development of novel biological or biotechnological management solutions urgently needed against rootworms.

zmsbt1 and zmsbt2, two new subtilisin-like serine proteases genes expressed in early maize kernel development.

MAIN CONCLUSION: Two subtilisin-like proteases show highly specific and complementary expression patterns in developing grains. These genes label the complete surface of the filial-maternal interface, suggesting a role in filial epithelial differentiation. The cereal endosperm is the most important source of nutrition and raw materials for mankind, as well as the storage compartment enabling initial growth of the germinating plantlets. The development of the different cell types in this tissue is regulated environmentally, genetically and epigenetically, resulting in the formation of top-bottom, adaxial-abaxial and surface-central axes. However, the mechanisms governing the interactions among the different inputs are mostly unknown. We have screened a kernel cDNA library for tissue-specific transcripts as initial step to identify genes relevant in cell differentiation. We report here on the isolation of two maize subtilisin-related genes that show grain-specific, surficial expression. zmsbt1 (Zea mays Subtilisin1) is expressed at the developing aleurone in a time-regulated manner, while zmsbt2 concentrates at the pedicel in front of the endosperm basal transfer layer. We have shown that their presence, early in the maize caryopsis development, is dependent on proper initial tissue determination, and have isolated their promoters to produce transgenic reporter lines that assist in the study of their regulation.

Two maize END-1 orthologs, BETL9 and BETL9like, are transcribed in a non-overlapping spatial pattern on the outer surface of the developing endosperm.

In the course of a project aimed to isolate transfer cells-specific genes in maize endosperm we have identified the BETL9 gene. BETL9 encodes for a small protein very similar in sequence to the product of the barley transfer cell-specific gene END-1. Both BETL9 and END-1 proteins are lipid transfer proteins, but their function is currently unknown. In situ hybridization analysis confirms that the BETL9 gene is exclusively transcribed in the basal endosperm transfer cell layer during seed development since 10 days after pollination. However, immunolocalization data indicates that the BETL9 protein accumulates in the maternal placento-chalaza cells located just beside the transfer cell layer. This suggests that the BETL9 protein should be transported to the maternal side to exert its, still unknown, function. In addition, we have identified a second maize gene very similar in sequence to BETL9 and we have named it BETL9like. In situ hybridization shows that BETL9like is also specifically transcribed in the developing maize endosperm within the same time frame that BETL9, but in this case it is exclusively expressed in the aleurone cell layer. Consequently, the BETL9 and BETL9like genes are transcribed in a non-overlapping pattern on the outer surface of the maize endosperm. The BETL9 and BETL9like promoter sequences, fused to the GUS reporter gene, accurately reflected the expression pattern observed for the genes in maize. Finally, we have identified in the Arabidopsis genome a set of four genes orthologous to BETL9 and BETL9like and analyzed the activity of their promoters in Arabidopsis transgenic plants carrying fusions of their promoter sequences to the GUS reporter. As in the case of the maize genes, the Arabidopsis orthologs showed highly complementary expression patterns.

Gene targeting in maize by somatic ectopic recombination.

Low transformation efficiency and high background of non-targeted events are major constraints to gene targeting in plants. We demonstrate here applicability in maize of a system that reduces the constraint from transformation efficiency. The system requires regenerable transformants in which all of the following elements are stably integrated in the genome: (i) donor DNA with the gene of interest adjacent to sequence for repair of a defective selectable marker, (ii) sequence encoding a rare-cutting endonuclease such as I-SceI, (iii) a target locus (TL) comprising the defective selectable marker and I-SceI cleavage site. Typically, this requires additional markers for the integration of the donor and target sequences, which may be assembled through cross-pollination of separate transformants. Inducible expression of I-SceI then cleaves the TL and facilitates homologous recombination, which is assayed by selection for the repaired marker. We used bar and gfp markers to identify assembled transformants, a dexamethasone-inducible I-SceI::GR protein, and selection for recombination events that restored an intact nptII. Applying this strategy to callus permitted the selection of recombination into the TL at a frequency of 0.085% per extracted immature embryo (29% of recombinants). Our results also indicate that excision of the donor locus (DL) through the use of flanking I-SceI cleavage sites may be unnecessary, and a source of unwanted repair events at the DL. The system allows production, from each assembled transformant, of many cells that subsequently can be treated to induce gene targeting. This may facilitate gene targeting in plant species for which transformation efficiencies are otherwise limiting.

Maternal control of nutrient allocation in plant seeds by genomic imprinting.

Imprinted genes are commonly expressed in mammalian placentas and in plant seed endosperms, where they exhibit preferential uniparental allelic expression. In mammals, imprinted genes directly regulate placental function and nutrient distribution from mother to fetus; however, none of the >60 imprinted genes thus far reported in plants have been demonstrated to play an equivalent role in regulating the flow of resources to the embryo. Here we show that imprinted Maternally expressed gene1 (Meg1) in maize is both necessary and sufficient for the establishment and differentiation of the endosperm nutrient transfer cells located at the mother:seed interface. Consistent with these findings, Meg1 also regulates maternal nutrient uptake, sucrose partitioning, and seed biomass yield. In addition, we generated an imprinted and nonimprinted synthetic Meg1 ((syn)Meg1) dosage series whereby increased dosage and absence of imprinting both resulted in an unequal investment of maternal resources into the endosperm. These findings highlight dosage regulation by genomic imprinting as being critical for maintaining a balanced distribution of maternal nutrients to filial tissues in plants, as in mammals. However, unlike in mammals, Meg1 is a maternally expressed imprinted gene that surprisingly acts to promote rather than restrict nutrient allocation to the offspring.

Duplicate maize Wrinkled1 transcription factors activate target genes involved in seed oil biosynthesis.

WRINKLED1 (WRI1), a key regulator of seed oil biosynthesis in Arabidopsis (Arabidopsis thaliana), was duplicated during the genome amplification of the cereal ancestor genome 90 million years ago. Both maize (Zea mays) coorthologs ZmWri1a and ZmWri1b show a strong transcriptional induction during the early filling stage of the embryo and complement the reduced fatty acid content of Arabidopsis wri1-4 seeds, suggesting conservation of molecular function. Overexpression of ZmWri1a not only increases the fatty acid content of the mature maize grain but also the content of certain amino acids, of several compounds involved in amino acid biosynthesis, and of two intermediates of the tricarboxylic acid cycle. Transcriptomic experiments identified 18 putative target genes of this transcription factor, 12 of which contain in their upstream regions an AW box, the cis-element bound by AtWRI1. In addition to functions related to late glycolysis and fatty acid biosynthesis in plastids, the target genes also have functions related to coenzyme A biosynthesis in mitochondria and the production of glycerol backbones for triacylglycerol biosynthesis in the cytoplasm. Interestingly, the higher seed oil content in ZmWri1a overexpression lines is not accompanied by a reduction in starch, thus opening possibilities for the use of the transgenic maize lines in breeding programs.

Bias due to withdrawal in long-term randomised trials in COPD: evidence from the TORCH study.

INTRODUCTION: Randomised controlled trials (RCTs) are considered the least biased method for evaluating drug efficacy and several large long-term RCTs in chronic obstructive pulmonary disease have been published. These usually include drugs with symptomatic benefits and have significant withdrawal rates. OBJECTIVES: We aimed at examining bias due to differential withdrawal in the Towards a Revolution in COPD Health (TORCH) trial. METHODS: We did an observational study nested in the TORCH trial, a placebo-controlled trial of salmeterol/fluticasone propionate combination (SFC) therapy in chronic obstructive pulmonary disease. We included 3057 patients randomly allocated to placebo or SFC in the analyses. We examined rates of withdrawal from the study and analysed change in effect parameters over time and in relation to withdrawal, as well as medication uptake after withdrawal. RESULTS: There was differential withdrawal with a significantly higher withdrawal rate from the group allocated to placebo than to SFC, 44% compared with 34%. Regardless of treatment group, withdrawal was associated with worse baseline lung function and more frequent exacerbations, leading to selection of a study population in better health than those originally recruited. As a result, annualized exacerbation rates in the first 6 months of the study compared with the last 6 months of the study decreased from 6.8 to 0.9 in the placebo group and from 3.0 to 0.8 in the SFC group. Also, use of medications under test in the study was frequent in patients after withdrawal. CONCLUSION: Significant bias may occur in long-term RCTs of registered medications with symptomatic benefits as a result of differential withdrawal.

Atypical response regulators expressed in the maize endosperm transfer cells link canonical two component systems and seed biology.

BACKGROUND: Two component systems (TCS) are phosphotransfer-based signal transduction pathways first discovered in bacteria, where they perform most of the sensing tasks. They present a highly modular structure, comprising a receptor with histidine kinase activity and a response regulator which regulates gene expression or interacts with other cell components. A more complex framework is usually found in plants and fungi, in which a third component transfers the phosphate group from the receptor to the response regulator. They play a central role in cytokinin mediated functions in plants, affecting processes such as meristem growth, phyllotaxy, seed development, leaf senescence or tissue differentiation. We have previously reported the expression and cellular localization of a type A response regulator, ZmTCRR-1, in the transfer cells of the maize seed, a tissue critical for seed filling and development, and described its regulation by a tissue specific transcription factor. In this work we investigate the expression and localization of other components of the TCS signalling routes in the maize seed and initiate the characterization of their interactions. RESULTS: The discovery of a new type A response regulator, ZmTCRR-2, specifically expressed in the transfer cells and controlled by a tissue specific transcription factor suggests a previously unknown role for TCS in the biology of transfer cells. We have characterized other canonical TCS molecules, including 6 histidine kinases and 3 phosphotransfer proteins, potentially involved in the atypical transduction pathway defined by ZmTCRR-1 and 2. We have identified potential upstream interactors for both proteins and shown that they both move into the developing endosperm. Furthermore, ZmTCRR-1 expression in an heterologous system (Arabidopsis thaliana) is directed to xylem parenchyma cells, probably involved in transport processes, one of the major roles attributed to the transfer cell layer. CONCLUSIONS: Our data prove the expression of the effector elements of a TCS route operating in the transfer cells under developmental control. Its possible role in integrating external signals with seed developmental processes is discussed.

The maize transcription factor myb-related protein-1 is a key regulator of the differentiation of transfer cells.

Transfer cells are highly modified plant cells specialized in the transport of solutes. They differentiate at many plant exchange surfaces, including phloem loading and unloading zones such as those present in the sink organs and seeds. In maize (Zea mays) seeds, transfer cells are located at the base of the endosperm. It is currently unknown how apical-basal polarity is established or why the peripheral cells at the base of the endosperm differentiate into transfer instead of aleurone cells. Here, we show that in epidermal cells committed to develop into aleurone cells, the ectopic expression of the transfer cell-specific transcriptional activator Myb-Related Protein-1 (MRP-1) is sufficient to temporarily transform them into transfer cells. These transformed cells acquire distinct transfer cell features, such as cell wall ingrowths and an elongated shape. In addition, they express a number of MRP-1 target genes presumably involved in defense. We also show that the expression of MRP-1 is needed to maintain the transfer cell phenotype. Later in development, an observed reduction in the ectopic expression of MRP-1 was followed by the reversion of the transformed cells, which then acquire aleurone cell features.

Characterisation of a new reporter system allowing high throughput in planta screening for recombination events before and after controlled DNA double strand break induction.

DNA double strand breaks (DSBs) are created either by DNA damaging reagents or in a programmed manner, for example during meiosis. Homologous recombination (HR) can be used to repair DSBs, a process vital both for cell survival and for genetic rearrangement during meiosis. In order to easily quantify this mechanism, a new HR reporter gene that is suitable for the detection of rare recombination events in high-throughput screens was developed in Arabidopsis thaliana. This reporter, pPNP, is composed of two mutated Pat genes and has also one restriction site for the meganuclease I-SceI. A functional Pat gene can be reconstituted by an HR event giving plants which are resistant to the herbicide glufosinate. The basal frequency of intra-chromosomal recombination is very low (10(-5)) and can be strongly increased by the expression of I-SceI which creates a DSB. Expression of I-SceI under the control of the 35S CaMV promoter dramatically increases HR frequency (10,000 fold); however the measured recombinant events are in majority somatic. In contrast only germinal recombination events were measured when the meganuclease was expressed from a floral-specific promoter. Finally, the reporter was used to test a dexamethasone inducible I-SceI which could produce up to 200x more HR events after induction. This novel inducible I-SceI should be useful in fundamental studies of the mechanism of repair of DSBs and for biotechnological applications.

Highly efficient Agrobacterium-mediated transformation of wheat via in planta inoculation.

This chapter details a reproducible method for the transformation of spring wheat using Agrobacterium tumefaciens via the direct inoculation of bacteria into immature seeds in planta as described in patent WO 00/63398 (1). Transformation efficiencies from 1 to 30% have been obtained and average efficiencies of at least 5% are routinely achieved. Regenerated plants are phenotypically normal with 30-50% of transformation events carrying introduced genes at single insertion sites, a higher rate than is typically reported for transgenic plants produced using biolistic transformation methods.

The promoter of ZmMRP-1, a maize transfer cell-specific transcriptional activator, is induced at solute exchange surfaces and responds to transport demands.

Transfer cells have specializations that facilitate the transport of solutes across plant exchange surfaces. ZmMRP-1 is a maize (Zea mays) endosperm transfer cell-specific transcriptional activator that plays a central role in the regulatory pathways controlling transfer cell differentiation and function. The present work investigates the signals controlling the expression of ZmMRP-1 through the production of transgenic lines of maize, Arabidopsis, tobacco and barley containing ZmMRP-1promoter:GUS reporter constructs. The GUS signal predominantly appeared in regions of active transport between source and sink tissues, including nematode-induced feeding structures and at sites of vascular connection between developing organs and the main plant vasculature. In those cases, promoter induction was associated with the initial developmental stages of transport structures. Significantly, transfer cells also differentiated in these regions suggesting that, independent of species, location or morphological features, transfer cells might differentiate in a similar way under the influence of conserved induction signals. In planta and yeast experiments showed that the promoter activity is modulated by carbohydrates, glucose being the most effective inducer.

empty pericarp4 encodes a mitochondrion-targeted pentatricopeptide repeat protein necessary for seed development and plant growth in maize.

The pentatricopeptide repeat (PPR) family represents one of the largest gene families in plants, with >440 members annotated in Arabidopsis thaliana. PPR proteins are thought to have a major role in the regulation of posttranscriptional processes in organelles. Recent studies have shown that Arabidopsis PPR proteins play an essential, nonredundant role during embryogenesis. Here, we demonstrate that mutations in empty pericarp4 (emp4), a maize (Zea mays) PPR-encoding gene, confer a seed-lethal phenotype. Mutant endosperms are severely impaired, with highly irregular differentiation of transfer cells in the nutrient-importing basal endosperm. Analysis of homozygous mutant plants generated from embryo-rescue experiments indicated that emp4 also affects general plant growth. The emp4-1 mutation was identified in an active Mutator (Mu) population, and cosegregation analysis revealed that it arose from a Mu3 element insertion. Evidence of emp4 molecular cloning was provided by the isolation of four additional emp4 alleles obtained by a reverse genetics approach. emp4 encodes a novel type of PPR protein of 614 amino acids. EMP4 contains nine 35-amino acid PPR motifs and an N-terminal mitochondrion-targeted sequence peptide, which was confirmed by a translational EMP4-green fluorescent protein fusion that localized to mitochondria. Molecular analyses further suggest that EMP4 is necessary to regulate the correct expression of a small subset of mitochondrial transcripts in the endosperm.